Research
Cite this article: Labandeira CC et al. 2016
The evolutionary convergence of mid-Mesozoic
lacewings and Cenozoic butterflies.
Proc. R. Soc. B 283: 20152893.
http://dx.doi.org/10.1098/rspb.2015.2893
developmental biology
e-mail: rendong@mail.cnu.edu.cn
Conrad C. Labandeira
Electronic supplementary material is available
The evolutionary convergence of
mid-Mesozoic lacewings and
2,8 9,10,11 1 3,12at http://dx.doi.org/10.1098/rspb.2015.2893 or
via http://rspb.royalsocietypublishing.org.e-mail: labandec@si.eduKeywords:
angiosperms, gymnosperms, Kalligrammatidae,
Papilionoidea, tubular proboscis, wing eyespots
Authors for correspondence:
Dong RenReceived: 2 December 2015
Accepted: 12 January 2016
Subject Areas:
palaeontology, evolution,rspb.royalsocietypublishing.orgCarol L. Hotton , Anto´nia Monteiro , Yong-Jie Wang , Yulia Goreva ,
ChungKun Shih1,2, Sandra Siljestro¨m3,13,14, Tim R. Rose3, David L. Dilcher15
and Dong Ren1
1College of Life Sciences, Capital Normal University, Beijing 100048, People’s Republic of China
2Department of Paleobiology, National Museum of Natural History, Smithsonian Institution, Washington,
DC 20013, USA
3Department of Mineral Sciences, National Museum of Natural History, Smithsonian Institution, Washington,
DC 20013, USA
4Department of Entomology and BEES Program, University of Maryland, College Park, MD 20742, USA
5State Key Laboratory of Biocontrol, Key Laboratory of Biodiversity Dynamics and Conservation of Guangdong
Higher Education Institute, College of Ecology and Evolution, School of Life Sciences, Sun Yat-sen University,
Guangzhou 510275, People’s Republic of China
6Geoscience Museum, Shijiazhuang University of Economics, Shijiazhuang 050031, People’s Republic of China
7Department of Crop and Agroenvironmental Sciences, University of Puerto Rico, Mayagu¨ez, PR 00681, USA
8National Centre for Biotechnology Information, National Library of Medicine, Bethesda, MD 20892, USA
9Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06511, USA
10Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore
11Yale-NUS College, Singapore 138614, Singapore
12Jet Propulsion Laboratory, National Aeronautics and Space Administration, Pasadena, CA 91125, USA
13Department of Chemistry, Materials and Surfaces, SP Technical Research Institute of Sweden,
Bora˚s 51115, Sweden
14Geophysical Laboratory, Carnegie Institution of Washington, Washington, DC 20015, USA
15Departments of Geology and Biology, Indiana University, Bloomington, IN 47405, USA
Mid-Mesozoic kalligrammatid lacewings (Neuroptera) entered the fossil record
165 million years ago (Ma) and disappeared 45 Ma later. Extant papilionoid
butterflies (Lepidoptera) probably originated 80–70 Ma, long after kalligram-
matids became extinct. Although poor preservation of kalligrammatid fossils
previously prevented their detailed morphological and ecological characteriz-
ation, we examine new, well-preserved, kalligrammatid fossils from Middle
Jurassic and Early Cretaceous sites in northeastern China to unravel a sur-
prising array of similar morphological and ecological features in these two,
unrelated clades. We used polarized light and epifluorescence photography,
SEM imaging, energy dispersive spectrometry and time-of-flight secondary
ion mass spectrometry to examine kalligrammatid fossils and their en-
vironment. We mapped the evolution of specific traits onto a kalligrammatid
phylogeny and discovered that these extinct lacewings convergently evolved
wing eyespots that possibly contained melanin, and wing scales, elongate tub-
ular proboscides, similar feeding styles, and seed–plant associations, similar to
butterflies. Long-proboscid kalligrammatid lacewings lived in ecosystemswith
gymnosperm–insect relationships and likely accessed bennettitalean pollina-
tion drops and pollen. This system later was replaced by mid-Cretaceous
angiosperms and their insect pollinators.
1. Introduction
Lepidoptera and Neuroptera are members of two basal clades of Holometabola
that separated ca 320 million years ago (Ma) during the mid-Carboniferous
& 2016 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
author and source are credited.Cenozoic butterflies
Conrad C. Labandeira1,2,4, Qiang Yang1,5,6, Jorge A. Santiago-Blay2,7,
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283:2015289
2(b)
(a)
(e)
(g)[1,2]. Although butterflies (Lepidoptera; Papilionoidea) are
perhaps the most iconic group of insect pollinators [3], their
earliest definitive fossils occur at the Palaeocene–Eocene
boundary, 56 Ma [3]. Molecular studies of various family
level ranks [4,5] suggest an earlier, Late Cretaceous origin
at ca 80–70 Ma [5,6], considerably after the mid-Cretaceous
(125–100 Ma) angiosperm radiation [7]. Butterflies are
characterized by a distinctive ensemble of traits, such as
diurnal behaviour, tubular (siphonate) mouthparts, wing
eyespot patterns and wing scales [3,8,9]. These features
appeared at the origin of the clade, allowing butterflies inti-
mate association with more derived angiosperms during
the Late Cretaceous and Palaeogene (80–23 Ma), and led to
the coevolution and diversification of both groups [5,10].
Was this stereotypical assembly of butterfly features a one-
time innovation uniquely associated with angiosperms? Or
did the butterfly character-suite evolve in unrelated insect
lineages with earlier gymnosperms? Here, we report on a dis-
tinctive clade of butterfly-like insects, Kalligrammatidae
( f ) (l)
(m) (n) (o)
Figure 1. Kalligrammatid structural diversity. Specimens are from the late-Middle Jura
stan; and mid-Early Cretaceous Yixian Fm. (YIX), China (electronic supplementary ma
[11]. Arrows indicate proboscis tips. (a) Kalligramma circularia (JIU); (b) Affinigramma
(e) Oregramma aureolusa (YIX); ( f ) Ithigramma multinervia (YIX); (g) Abrigramma ca
cebrosa (YIX). (i– k) Lateral views of ovipositor structure in O. illecebrosa above: (i) int
and (k) lateral valve pairs. (l–q): five kalligrammatid wing eyespot and spot types det
1 wing eyespot with two outer rings and ca 15 contiguous ocules surrounding a centr
outer ring, light-hued inner area, and uninterrupted, pigmented central disc with sur
eyespot similar to (M) (Kallihemerobius feroculus, JIU); (o) Type 3 wing eyespot with a
central disc (Ithigramma multinervia, YIX); (p) Type 4 wing eyespot contains a few oc
and surrounding, dark outermost ring (K. circularia, JIU); and (q) Type 5 wing spot o
bars: solid, 10 mm; striped, 1 mm.(c) (d )
(h)
(i)(Neuroptera), and explore their biological convergence
with Papilionoidea.
Kalligrammatidae, or kalligrammatid lacewings
(figure 1a–i), are an enigmatic, almost entirely Eurasian
[11–13], mid-Mesozoic, holometabolous clade of large,
robust-bodied Neuroptera (lacewings). Kalligrammatids had
large wingspans, up to ca 160 mm [12], and are among the
largest and most conspicuous of mid-Mesozoic insects (elec-
tronic supplementary material, table S1). Kalligrammatids
were tentatively associated with seed plants [14–16], despite
their almost unknown mouthpart and ovipositor structures
[16]. Within Neuroptera, the Kalligrammatidae are included
within Myrmeleontiformia [17–19], a major clade that encom-
passes extant antlions, owlflies, silky-winged lacewings
(Psychopsidae), and spoon and thread-winged lacewings
(Nemopteridae) [20,21]. The Nemopteridae share significant
mouthpart and feeding similarities [21,22] with the Kalligram-
matidae whereas the Psychopsidae possess similar wing
features [16].
( j) (k)
(p) (q)
ssic Jiulongshan Fm. (JIU), China; Late Jurassic Karabastau Fm. (KAR), Kazakh-
terial, tables S2 and S3). At (a– i) are nine species showing general habitus
myrioneura (JIU); (c) A. myrioneura (JIU); (d ) Kallihemerobius feroculus (JIU);
lophleba (JIU); (h) Kalligramma brachyrhyncha (JIU); and (i) Oregramma ille-
act specimen; ( j ) complete ovipositor and posteriormost abdominal segments;
ailed in figures 2 and 3; electronic supplementary material, figure S1. (l ) Type
al pigmented disc (O. illecebrosa, YIX); (m) Type 2 wing eyespot with a single
rounding, non-contiguous ocules (Kallihemerobius almacellus, JIU); (n) Type 2
light-hued circular area and a few, variably sized ocules in a darkly pigmented
ules and others surrounding a pigmented central disc, a light-hued inner area
f a circular, pigmented central disc (Kallihemerobius aciedentatus, JIU). Scale
3
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plex
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283:20152893
3125
135
145
155
165
175
wing eyespo
1
3
5
2
4
6
Jiulongshan Fm.
Uda Fm.
Karabastau Fm.
Solnhofen Fm.
Yixian
Zaza Fm.
Khuduk Fms.
Anda, Shine
Waldhurst Clay
Sophogram-
matinae
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La
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not studied
formationMa Epo. Per.
wing eyespot
and spot types
Oregramma illecebrosa Kallihemerobius feroculus Kalligram
1 2 3
comAll examined kalligrammatid material originated from
fine-grained, often carbonaceous lake deposits in one Central
Asian and two East Asian localities (figure 2a; electronic sup-
plementary material, tables S2 and S3) [23–25]. The oldest
deposit is Daohugou, of the Jiulongshan Formation, Inner
Mongolia, fromnortheasternChina. This deposit is radiometri-
cally dated by 40K/40Ar at 164–165 Ma [26], a date supported
by slightly younger isotopic dates from overlying volcanic
deposits [26,27]. This date corresponds to the late Callovian
of the latest Middle Jurassic, using a standard international
timescale [28]. Diverse floras and the earliest known kalligram-
matid lacewings occur at Daohugou [23]. Karatau, the middle
deposit, is represented by the Mikhailovka and Aulie sites in
the Chiment Region of eastern Kazakhstan. The date of this
deposit, the Karabastau Formation [24], is uncertain within
the Late Jurassic, but floras [29], insects [30] and stratigraphy
[24] indicate a mid-Late Jurassic date, approximating 155 Ma.
The youngest deposit, the Yixian Formation of Liaoning
Province in northeastern China, consists of several sites separ-
ated in time and space. These sites encompass 40K/40Ar and
87Rb/87Sr dates ranging from 128.2 Ma low to 121.6 Ma high
Zygophlebius pseudo-
silveira
Bicyclus anynana
(Nymphalidae)
Idea lynceus
(Nymphalidae)
(b)
(g) (h)
(c)
Figure 2. Phylogenetic context of wing spots and eyespots in mid-Mesozoic kalligram
material, text S3). The best preserved fossil material was used for this analysis. (a)
plementary material, table S2), with right forewing eyespot/spot condition mapped on
spot type symbols are at upper-left; crosses are eyespot/spot absences. (b–g) Examp
grammatidae (b– f ), and modern Psychopsidae (g). These taxa correspond to a Type
two Type 5 double spots ( f ) matched by two spots in modern psychopsid (red arrow
Lepidoptera in (h– k), of butterfly species with Type 6 eyespots (h) and multiple T
butterfly eyespot (k), showing pigmentation similar to Type 2 and 3 eyespots (b),
Scale bars: solid, 10 mm; striped, 1 mm.complex
wing eyespot
K
al
lig
ra
m
m
in
a
An
ga
ro
gr
a
m
m
a
K
al
lig
ra
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St
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Aff
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Si
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m
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a
Kalligrammatinae Kallihemerobiinae
wing spots and eyespots
wing scales
siphonate proboscis
hyrhyncha Affingramma myrioneura Kallihemerobius acieden-
tatus4 5
(a)in the formation, with most material collected from the Jian-
shangou beds dated at ca 125 Ma [27,31], the date used in
this report. Although contentious, Yixian dates are supported
by a variety of palaeobiological evidence [27,32], buttressed
by pollen studies [33] linked to a distinctive megaflora in the
lower part of the unit [34]. Claims of a Late Jurassic age for
Yixian fossils represent range extensions of Early Cretaceous
lineages downward into the Late Jurassic [31]. The last
known kalligrammatid lacewing occurs in the upper Crato
Formation of northeastern Brazil, ca 120 Ma [13].
Lake deposits such as the Jiulongshan, Karabastau and
Yixian formations typically preserve plants and insects that
reveal surface details [23,30,31]. Frequently, resolution of such
features extends to colour patterns (figures 1a–i,l–q and
3e–g,i,k; electronic supplementary material, figure S2), gross
(figure1), todetailedmouthpart structure (electronic supplemen-
tary material, figures S1, S4 and S5), micromorphological details
of wing and mouthpart scales (figure 3a,b,h,j,l–p; electronic
supplementary material, figures S4 and S5), and reproduc-
tive plant features such as pollen (electronic supplementary
material, figures S1t, S5b and S6a– f) and fructifications that
Caligo telamonius
(Nymphalidae)
Pectinophora gossypiella
(Gelechiidae)
(i) ( j)
(d ) (e) ( f )
(k)
matids, with comparisons to modern lepidopterans (electronic supplementary
Most parsimonious tree of Kalligrammatidae phylogeny [11] (electronic sup-
to terminal clades and likely wing spot and eyespot origins. Wing eyespot and
les of right forewings with wing eyespots or spots from mid-Mesozoic Kalli-
1 eyespot (b), Type 2 eyespot (c), Type 3 eyespot (d ), Type 4 eyespot (e) and
s) in (g). Kalligrammatid wing eyespots and spots are compared to modern
ype 5 spots (i); moth lacking wing spots or eyespots ( j ); and modern owl
indicated by arrow pointing to an ocule series and longitudinal wing vein.
(d )
rspb.royalsocietypublish
4(c)(b)(a)reveal internal structures (electronic supplementary material,
figure 6g–i) that extends previous studies [34–37].
2. Material and methods
Theelectronic supplementarymaterialdocuments thegeneralmeth-
odological approaches and specific experimental procedures used in
(e)
(h)
(i) ( j)
(k)
(l)
(m)
(n) (o)
( f )
Figure 3. (Caption opposite.)six substudies that buttress our account of ultrastructure and mor-
phology of Mesozoic kalligrammatid lacewings. These studies are
(i) kalligrammatidmouthpart structure; (ii) an analysis of pigmenta-
tion within wing eyespots; (iii) geochemical analyses of opaque
plugs trapped within the food canal of a tubular proboscis; (iv, v)
two analyses on pollen occurring adjacent mouthpart contact sur-
faces; and (vi) taxonomic characterization of pollen in sedimentary
matrices adjacent kalligrammatid specimens. We also provide
(g)
(p)
ing.org
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283:20152893
ot type
and th
wing, d
(c) and
overlay
cence m
flatten
itish h
scale
k) from
ales, ea
lacking
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ctronic
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5documentation of kalligrammatid mouthpart morphology. The
techniques contributing to these six substudies are briefly outlined
below; details of instrumentation and equipment that were used,
specific imaging procedures and the protocol for geochemical
analyses are provided in the electronic supplementary material.
(a) Specimen imaging
Light, epifluorescence and scanning electron microscopy (SEM)
were used to closely examine a variety of kalligrammatid features
from gross structure to micromorphology. Structures as miniscule
and delicate as setae, wing scales, wing eyespot ocules and pollen
grains were captured by microscopic imaging techniques, includ-
ing the backscattering function linked to SEM imaging. Camera
lucida drawings were made (electronic supplementary material,
figure S1) to establish the most highly resolved scale available,
and included shape, size, surface features and inter-element
relationships of siphonate mouthpart structure.
(b) Geochemical analyses
The heads, mouthparts, wing scales and eyespots of several
specimens were intensely investigated by electron dispersion
spectroscopy (EDS) linked to an environmental chamber SEM
(electronic supplementary material, figure S2), also time-of-flight
secondary ion mass spectrometry (ToF-SIMS, electronic supple-
mentary material, figure S3) [38]. The latter technique produced
intriguing results regarding eyespot pigmentation, and several
EDS analyses characterized a structureless plug within the probos-
cis food canal of one specimen (electronic supplementarymaterial,
figure S4e– j ). Pollen was detected adjacent vestigial but highly
setose mandibulate mouthparts of a second specimen (electro-
nic supplementary material, figure S4a–d). Two morphotypes
Figure 3. (Opposite.) Microstructure of three kalligrammatid forewing eyesp
retains a broken scale base in cross-section of four lower (bottom arrows)
type receives distinctive flat scales on major veins present elsewhere on the
and eight ribs terminally on Kalligramma sp. (JIU). For comparison of (b), at
othidae) (electronic supplementary material, text S2), in a SEM at left (c) and
(e) Light photograph showing eyespot pigmentation pattern, with epifluores
ocules (g), each in a wing compartment surrounded by minor veins bearing
of a Type 2 eyespot of Kallihemerobius almacellus (JIU), showing seven wh
shows smaller empty scale sockets in interveinal areas and occasional larger
enlarged at (a). (k–n) and ( p) A light photograph of a Type 1 eyespot (
by whitish ocules and two dark outer rings. (l ) SEM detail of four curved sc
that lack scales. (m) Nearby scales. (n) Field of clumped scales on a wing region
displaying a ridged structure. Eyespot ocule at (o), from Kallihemerobius acieden
central-disc pigmented regions, bearing scales socketed on major veins. See ele
striped, 1 mm; dotted, 10 mm.of elongate cuticular scaleswere imaged from themouthparts, par-
ticularly the maxillary palps, of another specimen using a variety
of techniques that included SEM imaging (electronic supplementary
material, figure S5). Wing eyespot pigmentation was detected by
EDS by enhanced carbon concentrations that were intrinsic to the
eyespot centre and absent from other regions such as the eyespot
ocules, other body regions and adjacent rock matrix.
(c) Pollen study
Most sedimentarymatrices adjacent to the specimens thatwere acid
macerated failed to preserve pollen, attributable to the oxidized
condition of the encompassing rock. The matrix of one specimen,
however, provided a well-preserved spectrum of pollen in
macerated residues that were mounted on microscope slides for
characterization. The resulting pollen was consistent not only
with the known megaflora described from the same deposit
but also provided common and rare entomophilous pollen taxa
(electronic supplementary material, figure S6a– f ).3. Results
Recently, a comprehensive phylogenetic analyses of 30
wing (28 of 30), ovipositor and mouthpart characters
for 17 kalligrammatid genera and four outgroups resulted
in a single best-supported tree [11] (figure 2a). The phylo-
geny grouped the genera into five distinct clades, three of
which are new subfamilies [11] (figure 2a; electronic sup-
plementary material, table S1). The basalmost clade,
Sophogrammatinae, represents the plesiomorphic kalli-
grammatid condition of mandibulate mouthparts and the
absence of wing spots, eyespots, and scales. The four derived
clades include Kalligrammatinae, consisting of the speciose
Kalligramma and four related genera, and Kallihemerobiinae
with six genera. Meioneurinae comprises the sole genus
Meioneurites [16], which has a sister-group relationship
to Oregrammatinae, the latter consisting of three genera,
including probably the most derived genus, Oregramma.
Higher-level relationships within Kalligrammatidae are:
Sophogrammatinaeþ f[(Meioneurinae)þ (Oregrammatinae)]þ
[(Kalligrammatinae)þ (Kallihemerobiinae)]g.
In forewings, kalligrammatid eyespots and spots typically
are deployed on the upper surface midway to two-thirds of
the proximal-to-distal wing length, centred between two
major branches of the radial vein system. Six distinctive types
of forewing eyespots or spots occur on most species of the
four derived kalligrammatid clades, occurrences previously
known from some taxa [11,15], but not others [12]. The basal-
most clade has no wing spots or eyespots (figure 2a), as do
almost all modern neuropterans (figure 2g) [19]. There are
s and their cuticular scales. (a) Kalligrammatid ellipsoidal wing-scale socket
ree upper (top arrows) ribs, enlarged from upper-right of ( j ). This socket
epicted as an overlay drawing in (b), showing four longitudinal ribs basally
(d ) is a foreleg scale of the modern neuropteran Lomomyia squamosa (Ber-
drawing at right (d ). (e–h) A Type 4 eyespot of Kalligramma circularia (JIU).
icroscopy revealing a differently pigmented ocule ( f ), and three additional
ed, four-ribbed scales, four shown in the SEM at (h). (i) Light photograph
ued ocules surrounding a central pigmented disc, the boundary (template)
sockets on veins in the SEM at ( j ). Large wing-scale socket at upper-right
Oregramma illecebrosa (YIX), with dark pigmented central disc surrounded
ch socketed on a longitudinal vein; black arrows indicate alternating sockets
veins and eyespots and a fascicle of eight, large, detached scales in ( p), each
IU), shows a regular array of interveinal scale sockets, structurally distinct from
supplementary material, table S2 for specimen data; scale bars: solid, 10 mm;four eyespot types, each consisting of distinctive, differentially
pigmented rings surrounding a central pigmented disc with
small, whitish, oval-shaped ocules (Types 1–4; figures 1l–p
and 2b–e,h,k; electronic supplementary material, figure S2).
In addition, there are taxa with two simple spots, consisting
of a round, dark patch lacking concentric rings (Type 5;
figures 1q and 2f ). Eyespots of Type 6 were not mapped onto
the phylogeny, as wing characters of one Kalligramma sp.
were insufficiently preserved for inclusion in phylogenetic
analyses. In Type 1 eyespots, a second ring of dark pig-
mentation occur relative to single ringed Types 2–4 and 6
(figures 1l and 2b).
Forewing eyespot and spot types (figure 1l–q) were
mapped onto our best-supported tree (figure 2a), revealing
major patterns. In all outgroup taxa and the basalmost kalli-
grammatid clade of Sophogrammatinae, eyespots and spots
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6were absent. The evolution of spots and eyespots likely origi-
nated early within the kalligrammatid clade, in the sister
lineage to Sophogrammatinae (figure 2a). The four kalligram-
matid clades derived from this lineage exhibit a variety of
spot and eyespot patterns and absences. The most complex
eyespot type occurs late in three separate lineages, within
Oregrammatinae (Type 1; figure 1l; electronic supplementary
material, figure S2), Kallihemerobiinae and Kalligrammatinae
(figure 2a), suggesting that these eyespots derive from sim-
pler ones, a transition that likely happened multiple times.
In addition, multiple simple spots were converted to single
eyespots in several lineages. These patterns are similar to con-
vergent changes conventionally proposed for nymphalid
butterflies in modern Lepidoptera (figure 2h,i,k) [8,39,40].
Changes include transitions frommoth taxa possessing mono-
chromatic wings lacking differential pigmentation (figure 2j ),
to basal nymphalid taxa with simple repeated spots, such
as Idea lynceus (figure 2i), to more elaborate and indivi-
dualized eyespot patterns of Bicyclus anynana with multiple
colour rings (figure 2h) [8,39]. The deployment of a spot of
monochromatic pigment between two major veins in basal
Kallihemerobiinae, Kalligrammatinae and Oregrammatinae
(figure 2a,f) has convergently re-evolved in modern, distantly
related Psychopsidae (figure 2g) and Nemopteridae [19].
Another point of convergence is the possible presence of
melanin in wing eyespot centres as indicated by our EDS
carbon (electronic supplementary material, figure S2) and
ToF-SIMS (electronic supplementary material, figure S3) sub-
studies. SEM examination of the eyespots using EDS revealed
a significant increase in carbon content within black eyespot
centres, whereas the central white pupil was completely
devoid of carbon. In the ToF-SIMS analysis, the eumelanin
presence was indicated by comparison of the spectrum from
the dark eyespot pupil with the spectrum of a modern eumela-
nin standard. Owing to dissimilarities in the intensity of the
organic peaks, similar to what has been found in other studies
[41,42], the possibility of an alternative carbon source cannot be
excluded. Unlike melanin preserved in many animals, where it
occurs in rod-shaped specialized cells [43], insects lack such
cells and melanin is diffused throughout the cuticle [44]. The
relative abundances of carbon and the possible presence of
melanin found in differently coloured regions of kalligramma-
tid eyespots could match the pigment distribution in many
nymphalid eyespot patterns [39]. The muted response of
carbon-rich material in kalligrammatid eyespots could mimic
the nymphalid condition, as scales in an eyespot centre often
are devoid ofmelanin and reflect all lightwavelengths, appear-
ing white [45], whereas black scales encircling the eyespot
centre contain melanin [46].
Wing scales are another convergent feature occurring in
Kalligrammatidae and modern Lepidoptera, although there
are differences in detail. The basalmost clade, Sophogrammati-
nae, lacked wing scales, as do virtually all other modern, major
neuropteran lineages (figure 3c,d). The four derived kalligram-
matid clades bore two types of wing scales. The first type were
large scales with a flattened, elongate-spatulate shape socketed
on major veins and possessing three to four longitudinal
ribs, increasing to six to eight ribs at the distal wider end
(figure 3a,b,j,p; electronic supplementary material, figure S5a).
The second scale typewere small, short scales that were basally
broad but tapered, bearing four or fewer longitudinal ribs, and
originating from smaller sockets on areas between the major
veins (figure 3h,j,l–o; electronic supplementary material,figure S3d). This distribution indicates wing scales originated
de novo among early Kalligrammatidae, after separation
from Sophogrammatinae (figure 2a). By comparison, in extant
Lepidoptera, scales emerge predominantly from membrane
surfaces and minor veins, but often are absent on major veins
and larger cross-veins.
Mouthparts of kalligrammatid Neuroptera and papilio-
noid Lepidoptera offer another remarkable example of
convergent evolution. Kalligrammatid mouthparts evolved
from an ancestral mandibulate (chewing) state to a derived
long-proboscid (siphoning) state in which maxillary elements
were conjoined to form a tube (electronic supplementary
material, figure S1). This parallels the evolution of the probos-
cis in glossate Lepidoptera, which also originated from
mandible-bearing ancestors [47]. The kalligrammatid probos-
cis is present in all clades except basal Sophogrammatinae.
Rudimentary, mandible-bearing mouthparts were retained
in one long-proboscid specimen of Kallihemerobiinae (elec-
tronic supplementary material, figures S1t,u and S4), which
bore a much-reduced labium and specialized mandibles,
likely for pollen handling, indicated by adjacent pollen (elec-
tronic supplementary material, figure S1t). Rudimentary
mandibles parallel that of the extant Nemopteridae (elec-
tronic supplementary material, figure S1u), probable sister-
group of Kalligrammatidae [16], that currently have modified
mandibulate mouthparts attached to an anterior prolongation
of the head capsule for probing and nectaring flowers [9,19].
Many extinct and modern insects bear a long proboscis
[9,14,36,48], but the proboscides of more derived kalligramma-
tids bear a special resemblance to those of Lepidoptera [47].
The kalligrammatid proboscis was long (8–20 mm), flexible,
lacked stylets or other piercing structures, smooth or covered
with surface hairs, bracketed by multisegmented maxillary
palps, and its terminus typically rounded or truncate, resem-
bling the end of a thick straw (electronic supplementary
material, figure S1b,e)—all morphologies paralleling modern
Lepidoptera [49]. In addition, kalligrammatid proboscides
were longer and more robust, and thus differed from other
coexisting, long-proboscid lineages, such as the shorter and
more gracile, labellate pads borne by brachycerous flies
[35,48], and analogous pseudolabellae of aneuretopsychine
scorpionflies [36]. Suction forces were provided by one, per-
haps two, sucking pumps located in the frontal head region
(electronic supplementary material, figures S1 and S6i), mir-
roring those in Lepidoptera. The considerable mouthpart
variation in kalligrammatids, especially of the proboscis, is
comparable to modern Nymphalidae and other lepidop-
terans that probe for nectar and pollen at different floral
depths and resistance [5,9,39]. Some kalligrammatid taxa
bore thin and gracile proboscides (electronic supplementary
material, figure S1f,r,s), and likely probed into narrow and
shallow receptacles for ovular pollination drops and secretions
from pollen organs [7,14]. By contrast, the robust and compara-
tively longer mouthparts of other kalligrammatid taxa
(electronic supplementary material, figure S1i,j,p) were
likely suited to probe larger, sturdier reproductive structures
of Bennettitales, cycad-like plants contemporaneous with the
Kalligrammatidae.
Three substudies (electronic supplementary material)
explored the dietary range of kalligrammatid lacewings. The
first examination targeted an opaque plug trapped within the
food canal of a specimens’ proboscis (electronic supplementary
material, figure S4e– j), also seen under light microscopy
homologous. This indicates that wing-scale presence in the
rspb.royalsocietypublishing.org
Proc.R.Soc.B
283:20152893
7(electronic supplementary material, figure S1h), indicating a
bolus enriched in carbon and consistent with a diet of nectar-
like fluids. A second assessment found pollen associated with
themouthpartsof rudimentarymandibles inonespecimen (elec-
tronic supplementarymaterial, figures S1t,u and S4a–d). A third
evaluation identified typical mid-Mesozoic, Eurasian pollen
grains adjacent the maxillary palp base of another species
(electronic supplementary material, figure S5). An additional
substudy was a maceration of sedimentary matrix adjacent to
several insect bodies, with pollen consistent with published
megafloras from these localities (electronic supplementary
material, figure S6a– f). These substudies document a similarity
in feeding style and diet of kalligrammatid lacewings with
extant butterflies.
Likely hosts for Kalligrammatidae include cycads (Beania),
bennettitaleans (Williamsonia, Weltrichia) and caytonialeans
(Caytonia, Caytonianthus). Members of the bennettitaleans
and caytonialeans possessed the type of recessed ovules with
tubular access that would receive long, probing proboscides
of Kalligrammatidae [7,36,50–52]. SomeCheirolepidaceae pos-
sessed cone scales partially concealing deep funnels connected
to ovules [35]. Early angiosperms from the Yixian Formation
are delicate, aquatic, with small, nontubular flowers [34,53],
unlikely hosts for Kalligrammatidae. Larger gymnospermous
reproductive structures likely accommodated the more robust
spectrum of kalligrammatid siphoning proboscides (electronic
supplementary material, figure S1 and table S3).
Of all knownMesozoic gymnosperm groups, the bennetti-
talean family Williamsoniaceae most likely formed a close
pollinator mutualism with the Kalligrammatidae. Six lines of
evidence point to this inference. First, stoutly constructed and
elongate kalligrammatid proboscides match the deeply
placed fluids and pollen of bennettitaleans [7,50–52] (elec-
tronic supplementary material, figure S6g,h) better than other
co-occurring proboscid-bearing taxa [13]. At least two Late Jur-
assic to Early Cretaceous Eurasian ovulate organs,Williamsonia
bryonyae, and W. minima, had deep throats [50,52], and would
have accommodated the longer proboscis lengths of kalligram-
matid taxa, as would the Jiulongshan specimen (electronic
supplementary material, figure S6h). Second, Cycadopites and
other monosulcate pollen (electronic supplementary material,
figure S6c) are present in the Jiulongshan [54], Karabastau
[29,52] and Yixian [34] biotas, which also preserve diverse
Kalligrammatidae [11] and williamsoniaceous male (Weltrichia)
and female (Williamsonia) organs. Both taxa broadly coincide as
fossils during a 60 million-year period of the mid-Mesozoic.
Third, Weltrichia pollen organs (electronic supplementary
material, figure S6g) bore secretory glands [50,51], interpreted
as ‘nectaries’ [55], positioned below paired dehiscing pollen
sacs along the inner surfaces of clasping bract-like structures
[50,51,55]. Analogously, conspecific Williamsonia ovulate
organs (electronic supplementary material, figure S6h) pro-
duced pollination droplets [35,52]. These nutritional rewards
would have been lures for pollinator visits to male and female
organs. Fourth, cheirolepidaceous and other conifer pollen
occurred adjacent to the head and mouthparts on one kalli-
grammatid specimen (electronic supplementary material,
figure S1t) [35], suggesting seed–plant pollen consumption
and a predisposition for pollination [7], as pollen is often a
supplemental protein source in modern pollinating insects
[9,49]. Fifth, the presence of a curved, saw-like ovipositor
(figure 1i–k), homologous and similarly shaped to that of the
Dilaridae and used for inserting eggs into deep substratesKalligrammatidae and the absence in almost all other fossil
and modern neuropterans may be due to changes in deploy-
ment of the gene regulatory network within wings, rather
than independent origins of scales across Neuroptera.
There likely was an association between kalligrammatid
lacewings and coexisting gymnosperm seedplants.Diverse evi-
dence support this mid-Mesozoic association, including
gymnosperm pollen grains occurring in proximity to the
insects; mouthpart morphology designed for probing and
fluid feeding; carbon-rich compounds in a kalligrammatid[56], suggests that females sliced plant tissues for egg deposi-
tion and that their larvae consumed internal plant tissues,
explaining insect galleries in williamsoniaceous tissues [35]
and their expected occurrence in Early Cretaceous ambers
[38]. Sixth, placement of Weltrichia and Williamsonia organs on
separate parts of the same plant or on different conspecific
plants [50,51], indicates an outcrossing reproductive strategy.
For such functionally dioecious plants, wind may achieve
moderate levels of fertilization, but insects are significantly
more efficient [7].
4. Discussion and Conclusion
Several accounts [15,16]—some made nearly a century ago
[57,58]—have opined on the superficial similarity of poorly pre-
served kalligrammatid lacewings with modern butterflies. Such
analogies, however, were not based on detailed, ultrastructural,
micro- andmacromorphological, geochemical and palynological
evidence. In this study, a broad array of evidence is marshalled
to support structural convergence between mid-Mesozoic kalli-
grammatid lacewings and modern butterflies. This convergence
extends to possible melanin presence, simpler spots to complex
eyespots, wing scales, long-proboscid siphonate mouthparts,
feeding style similarities, and associations with seed plants.
These major convergences appeared twice in time and space,
presumably under similar selective pressures.
Our data allow for inferences regarding the ecology of
insect–predator antagonistic interactions. Similarities between
kalligrammatid eyespots and butterfly eyespots lie in the use
of concentric circles of pigmented cells to produce a conspicu-
ous and contrasting display. This pattern was used either for
predator intimidation or alternatively predator deflection to
the wings away from the core body in extinct kalligrammatids,
serving the same functions in butterflies [59,60]. Repeated evol-
ution of eyespots from simpler multiple spots arose during the
Middle Jurassic in Kalligrammatidae (figure 2a), closely paral-
leling Nymphalidae ca 110 Myr later [39]. An ecological
explanation for why multiple wing spots were replaced by
singlewing eyespots in Kalligrammatidae may be the eyespot’s
larger and more effective startle or deterrent signal [61].
Eyespots likely were used to dissuade or deflect attacks by pre-
dators such as early birds or small theropod dinosaurs [60,61]
or mantid insects [59].
Wing scales appeared in Middle Jurassic Kalligrammati-
dae and Early Palaeogene Lepidoptera. Previously, wing
scales were not documented on other fossil or modern neu-
ropterans. Our survey of NMNH Neuroptera (figure 3c,d )
found a single occurrence of scales on the forewings of one
genus of extant, unrelated Berothidae [62]. Although these
scales have differences in branching and number of ribs com-
pared to those of Kalligrammatidae (figure 3b), they likely are
ex
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rspb.royalsocietypublishing.org
Proc.R.Soc.B
283:20152893
8modifications that would accommodate long-proboscides
[7,14]. Varied fossil data suggest that the mid-Cretaceous
demise of many pre-existing gymnosperms led to extinction of
their diverse insect associates [14,30,63–65], includingKalligram-
matidae, during early angiosperm diversification. Intriguingly,
this clade was replaced by ecologically convergent butterflies ca
60 Myr later.
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Author contributions. D.
and D.R. designed
D.R. provided work
J.A.S.B. and C.C.L. p
T.R.R. and Y.G. wor
analyses; A.M. prov
scale data. C.L.H.
C.K.S., C.C.L. and
and S.S. conducted
provided macroflora
Competing interests. We
Funding. This work w
gram of China (973
Foundation of Chi
41272006 and 41372
Project (grant no. 20
(grant no. 2012T501
China (grant no. 201
(grant 5132008), Gre
Commission of Edu
Changjiang Scholars
(IRT13081), Natural
no. C2015403012),
Institutes of Health
Carbon Observatoryming the basis of this research and the details
ble in the electronic supplementary material
.
and Q.Y. prepared the fossil material. C.C.L.
research and wrote the paper. Q.Y., Y.-J.W.,
n systematics and phylogeny reconstruction.
vided wing eyespot and mouthpart analyses;
d on light microscopy, SEM, EDS and related
ed expertise on interpretation of eyespot and
ntified pollen and interpreted their context,
. worked on systematics and mimicry; Y.G.
e ToF-SIMS analysis; and D.L.D. and C.C.L.
nsights.
eclare we have no competing interests.
supported by the National Basic Research Pro-
gram) (grant 2012CB821906), National Science
(grant nos. 31230065, 31309105, 31372243,
), Beijing Municipal Commission of Education
07120), China Postdoctoral Science Foundation
), Doctoral Program of Higher Education of
108120005), Beijing Natural Science Foundation
Wall Scholar Project of the Beijing Municipal
tion (grant no. KZ201310028033), Program for
nd Innovative Research Teams at University
cience Foundation of Hebei Province (grant
tramural Research Program of the National
ibrary of Medicine, to C.L.H. and the Deep
Y.G. and S.S. S.S. also was supported through
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